On Sun, 3 May 1998, Petr Kuzmic (pk) wrote:
> claude aflalo (ca) wrote:
> > As a corollary, the
> > behavior of an immobilized enzyme observed locally should ideally be
> > identical to that in solution.
> pk:
> This probably implies a physical picture in which the immobilized enzyme is
> floating around attached to a -tether-. If I simply snip the tether, and then
> study a soluble form of the previously immobilized enzyme [as is often done],
> I should observe very little change in catalytic properties. However, if an
> enzyme is embedded in a multienzyme complex or partially burried in the
> membrane itself, I suppose that this must change the structure (tertiary, or
> even secondary) of the protein molecule. I supposed that under those
> circumstance the behavior of the solubilized and immobilized enzyme should be
> intrinsically different...
>ca:
Correct. My point was that the simple immobilization of an enzyme on an
inert support is often sufficient to dramatically alter its EFFECTIVE
behavior as observed by macroscopic measurements (e.g. v vs.[S] in the
medium) with no changes in INTRINSIC properties. For instance, an enzyme
entrapped in a bead will appear to use its substrate less efficiently
(higher apparent Km), but no change in kcat should be noted. This is
because the local [S] in the bead, responsible for the observed behavior,
is lower than [S] in the medium at steady state, due to diffusion
limitation (the local rate of catalysis by the concentrated enzyme in the
bead becomes high relative to diffusion). Nevertheless, a plot of v vs.
[S] in the bead (local) should remain identical to that obtained in
solution as long as the support is inert. The origin of the observed
variation in this case is purely dynamic (high ratio between the rate of
catalysis and that of diffusion; the latter becomes more limiting).
A more complex interaction of the enzyme with the support may be expected
to yield additional effects related to intrinsic properties of the enzyme
(kcat and/or Km). Interaction with reactants or effectors will affect in
addition their reactivity towards the enzyme.
ca:
> > Experimental measurements
> > indicate that by increasing hexokinase concentration added to the medium,
> > while bulk [ATP] detected by soluble luciferase becomes vanishingly small,
> > its local concentration (detected by localized luciferase) tends to a
> > non-zero, finite value which is not decreased further by additional
> > hexokinase. The results are consistent with a limiting mass transfer of
> > nucleotides (ATP and ADP) between bulk solution and the outer membrane of
> > mitochondria (upon which ATP appears and accumulates, while ADP disappears
> > and is depleted, relatively to bulk solution).
>pk:
> I think that is what Gary Nelsestuen is saying in
>> @article { Nels9781
> author = "G. L. Nelsestuen and M. B. Martinez",
> title = "Steady State Enzyme Velocities That Are Independent
> of [Enzyme]: An Important Behavior in Many Membrane
> and Particle-Bound States",
> journal = "Biochemistry",
> volume = "36", pages = "9081-9086", year = "1997" }
>> It makes perfect sense to me. I think that directly at the solid/liquid
> interface, the substrate and product molecules will be frozen in place by
> adsorption, so not even diffusion [much less than laminar flow, which is zero
> directly at the interface] can move them.
>ca:
I wouldn't say "frozen" which implies that they went "out of the system".
In fact the reactant are still available at the interface, but at a
concentration - or rather chemical activity - which may be different than
that in the bulk solution.
ca:
> > For instance, fixed negative
> > charges on the support should yield increased or decreased apparent Km's
> > for negatively or positively charged substrates, respectively, due to
> > their electrostatic partition (equilibrium) between the microenvironment
> > of the enzyme and the bulk solution, independently of - and in addition to
> > - diffusional resistance (steady state).
>pk:
> [I suppose that is just one example of how what I called 'adsorption' might happen.]
ca:
Right
ca:
>> > The interpretation of macroscopic (bulk) measurements of a localized
> > enzyme is difficult since the properties of the support (say a biological
> > membrane) are often poorly defined. It is however sensible that a change
> > in an apparent kinetic constant (say Ki) may be attributable to INHERENT
> > properties of the enzyme localized on a membrane.
pk:>
> Claude, have you (or anyone you are aware of) ever measured inhibition
> constants for an immbilized enzyme, then took the same or equivalent enzyme
> and repeated the study in solution?
>> -- Petr
ca:
Petr, I don't have data on this specific case. However, I think that you
can find some relevant citations in the following review (maybe in the
whole volume):
Goldstein, L. (1976) Methods Enzymol. 44, 397-443
I promise to have a look at it (I have to dig it out).
Nevertheless, the case of inhibitor will rely less on the simple dynamic
effect I mentionned above re. reactant concentrations. Indeed changes in
apparent Ki for an immobilized enzyme result from
1. changes in local concentration of the inhibitor (static, e.g. equilib.)
2. changes in local concentration of the reactants (dynamic, steady state)
3. changes in the local ratio between reactants and inhibitor.
This case is more complex to study...
Claude
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